Cell cultures is a tool to study biological and biochemical mechanisms involved in differentiation, cell survival and death; however, the use of mixed cultures has rarely explored. The aim of this study was to establish the conditions of a mixed culture (Neurons and astrocytes) from postnatal rat cells (P3) describing the morphometric characteristics of these cells and the general behavior of coculture in a time course of 6, 7, 8 and 9 days. Morphologic changes in neurons (MAP-2) and astrocytes (GFAP) with nuclear counterstain (Hoechst) were determined by immunocytochemistry and eight quantitative parameters for each linage were assessed by using Image-J 1.49 software (NIH). Cell density, perimeter, area of neurons and astrocytes increased on day 8 (p<0.0001) compared to day 6 from the mixed culture. Cell count, area, number and length of neurites (p>0.05) did not show differences. The ratio astrocytes / neurons was 1:1, without differences between days of culture. In conclusion, the mixed cell culture neuron-astrocyte under the conditions described in this study allow the morphometric characterization of those cells and constitute an important tool in studies of neuroprotection and neurotoxicity in nerve tissue in vitro.